![Silent cold-sensing neurons are activated by ciguatoxin-2. (A) Behavioural testing of the effect of intraplantar injection of 100 nM ciguatoxin-2 (P-CTX-2) on cold sensitivity n = 6 for sham vehicle (three males and three females) and n = 6 for P-CTX-2 (three males and three females). Means were compared by repeated measures two-way ANOVA followed by post hoc Sidak’s test. Error bars denote 95% confidence interval. (B) Example images and traces of a large-diameter neuron (Cell 1) that is basally cold-insensitive but begins to respond to cooling after treatment with P-CTX-2. [C(i)] Heat map showing the effect of P-CTX-2 on the number of neurons responding to a cold ice-water stimulus. n = 48 for vehicle and n = 196 for P-CTX-2. The bar corresponds to 15 s. [C(ii)] Summary of the change in the number of sensory neurons responding to each modality after treatment with P-CTX-2. (D) Histograms of cross-sectional area of all neurons responding to any cold stimulus in the naïve state (left, blue, n = 91) and after P-CTX-2 (right). For P-CTX-2, blue denotes basally responsive neurons that maintained their response to cold (n = 70) and red denotes the silent cold-sensing neurons that were unmasked after treatment (n = 136). The distribution of areas in the naïve state was fit by non-linear regression (least squares Gaussian; bin width is 80 µm2; mean = 212.4 µm2, SD 73.33 µm2). This model is plotted over the P-CTX-2 data to aid comparison with the dashed line denoting 3 SD from the mean. The different in the distribution of areas between groups was assessed by Kolmogorov-Smirnov test (P < 0.001). [E(i)] Box plot of the change in activation threshold of basally cold-sensitive neurons before and after treatment with vehicle (n = 35) or P-CTX (n = 8). [E(ii)] Box plot of the thermal activation threshold of all silent cold-sensing neurons unmasked by P-CTX-2 (n = 43) compared to all cold-sensing neurons recorded from naïve mice (n = 62). Medians were compared by Mann-Whitney test. (F) Quantification of the proportion of neurons responding ice-water that were also sensitive to either mechanical (i) or heat (ii) before and after treatment. Vehicle: npre = 36, npost = 43. P-CTX-2: npre = 69, npost = 174. [F(iii)] Comparison of the proportion of silent cold-sensing neurons that were responsive to other modalities before and after the induction of cold-sensitivity by P-CTX-2. n = 127. The proportion of polymodal neurons was compared using a χ2 test, and error bars denote 95% confidence intervals. For this experiment, 615 neurons responding to any stimulus either before or after treatment were recorded in 10 P-CTX-2-injected mice (four males and six females) and 193 cells were recorded from three vehicle-injected animals (two males and one female).](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/brain/144/6/10.1093_brain_awab086/1/awab086f3.jpeg?Expires=1749433098&Signature=PbEK3Xds4-8aqFo5uq-IMLQL7bD8VyiHuSBCjqxNwIAjZKvF4tkLnxt--QBSUZlVhk5Q4dHmvQAZ8LYa80UqpMUB46JplMCrVBJnZduo6ag2GWzcAejHno9uvZiTJWBOqhk5Ua40XI2DBnYmvdNeB~EVPZrqXgPMsIchcMS-AepFiJR6PHtUfSZwkN0lHxhlo~IC1M0M9FnkMQi3jXF6Un8-8o0toiAtyQG5hEmlliMEPufppFnfNRtzj5IK9abKsKiOtE1ym~6oz9-368YVvGMarxAfqyuD347gro~BVbp8OzNmxCbIV7e2dDzSaZJtSKOGuke0Z~ES6fj8pP2cJA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Silent cold-sensing neurons are activated by ciguatoxin-2. (A) Behavioural testing of the effect of intraplantar injection of 100 nM ciguatoxin-2 (P-CTX-2) on cold sensitivity n = 6 for sham vehicle (three males and three females) and n = 6 for P-CTX-2 (three males and three females). Means were compared by repeated measures two-way ANOVA followed by post hoc Sidak’s test. Error bars denote 95% confidence interval. (B) Example images and traces of a large-diameter neuron (Cell 1) that is basally cold-insensitive but begins to respond to cooling after treatment with P-CTX-2. [C(i)] Heat map showing the effect of P-CTX-2 on the number of neurons responding to a cold ice-water stimulus. n = 48 for vehicle and n = 196 for P-CTX-2. The bar corresponds to 15 s. [C(ii)] Summary of the change in the number of sensory neurons responding to each modality after treatment with P-CTX-2. (D) Histograms of cross-sectional area of all neurons responding to any cold stimulus in the naïve state (left, blue, n = 91) and after P-CTX-2 (right). For P-CTX-2, blue denotes basally responsive neurons that maintained their response to cold (n = 70) and red denotes the silent cold-sensing neurons that were unmasked after treatment (n = 136). The distribution of areas in the naïve state was fit by non-linear regression (least squares Gaussian; bin width is 80 µm2; mean = 212.4 µm2, SD 73.33 µm2). This model is plotted over the P-CTX-2 data to aid comparison with the dashed line denoting 3 SD from the mean. The different in the distribution of areas between groups was assessed by Kolmogorov-Smirnov test (P < 0.001). [E(i)] Box plot of the change in activation threshold of basally cold-sensitive neurons before and after treatment with vehicle (n = 35) or P-CTX (n = 8). [E(ii)] Box plot of the thermal activation threshold of all silent cold-sensing neurons unmasked by P-CTX-2 (n = 43) compared to all cold-sensing neurons recorded from naïve mice (n = 62). Medians were compared by Mann-Whitney test. (F) Quantification of the proportion of neurons responding ice-water that were also sensitive to either mechanical (i) or heat (ii) before and after treatment. Vehicle: npre = 36, npost = 43. P-CTX-2: npre = 69, npost = 174. [F(iii)] Comparison of the proportion of silent cold-sensing neurons that were responsive to other modalities before and after the induction of cold-sensitivity by P-CTX-2. n = 127. The proportion of polymodal neurons was compared using a χ2 test, and error bars denote 95% confidence intervals. For this experiment, 615 neurons responding to any stimulus either before or after treatment were recorded in 10 P-CTX-2-injected mice (four males and six females) and 193 cells were recorded from three vehicle-injected animals (two males and one female).
